PROJECT SUMMARY/ABSTRACT Diseases such as periodontitis, osteoporosis, and rheumatoid arthritis, are characterized by excessive bone resorption due to increased osteoclast activity. It is estimated that osteoporosis affects ~10 million Americans with another 19 million at risk. Periodontitis affects ~46% of the U.S. adults aged 30 years. Rheumatoid arthritis affects ~54.4 million U.S. adults - equating to about 25% of the population. The impact of these diseases in terms of social, psychological, and economic burden on individuals, communities, and health services is enormous. Despite these concerns, the molecular mechanisms contributing to dysregulated osteoclast function in these pathological conditions remain unclear, creating a critical knowledge gap in developing targeted interventions and therapeutic strategies. Our long-term goal is to identify and gain an in-depth understanding of the positive and negative regulators of osteoclast differentiation and utilize the gained knowledge to develop new interventional and therapeutic approaches for osteoclast-mediated disorders. Towards achieving our long-term goal, we have identified a novel mutation in the Interferon Regulatory Factor 8 (IRF8) gene that promotes increased osteoclast activity and susceptibility to a rare periodontal disease termed ?Multiple Idiopathic Cervical Root Resorption (MICRR). The current proposal takes a new direction to gain in-depth knowledge about IRF8 role in osteoclast regulation and periodontal homeostasis. To date, IRF8?s role in osteoclastogenesis has been studied using Irf8 gKO mice. However, the severely altered population and properties of hematopoietic stem cells in Irf8 gKO mice has affected the detailed analysis of IRF8 function in osteoclast precursors derived from a monocyte/macrophage lineage. To overcome this limitation, we have generated a myeloid cell-specific Irf8 conditional knockout (cKO). In this application, we will apply genetic approaches and use both in vitro cell culture systems and Irf8 cKO mouse model to investigate: 1) Importance of IRF8 in monocyte subset development (2) Osteoclastogenic potential of different monocyte subsets and how it is influenced by IRF8, and (3) Specific monocyte subsets contributing to tissue destruction in periodontitis. We will pursue these aims using an innovative combination of Irf8 cKO mouse model and techniques such next-generation sequencing, flow cytometry, and micro-CT. The proposed research is significant, because it will provide critical mechanistic insights into the regulation of complex networks of transcription factors that govern osteoclast differentiation. This work will develop foundational resources that will be used by other researchers for studying various bone disorders and inflammatory disorders that have known risk alleles in IRF8. Most importantly, the results will lay the groundwork to develop better treatment options for various osteoclast-mediated bone disorders, including periodontal disease.